656 VOLUME 17 NUMBER 6 JUNE 2016 NATURE IMMUNOLOGY The innate lymphoid cell (ILC) family orchestrates immunity, inflammation, metabolic homeostasis and tissue repair in multiple tissues, including intestine, liver, adipose, skin and lung 1,2 . Group 2 ILCs (ILC2s) are the dominant ILC population in human and murine lung, where they act as key initiators of allergen- and non- allergen-induced type 2 inflammation 1,3–5 and promote airway tissue repair 6 . ILC2s are activated by cell-extrinsic environmental cues such as the cytokines interleukin-25 (IL-25), IL-33 and thymic stromal lymphopioetin 1,2 . However, the cell-intrinsic pathways that regulate ILC2 effector function remain poorly characterized. In particular, regulation of cell metabolism is a critical determi- nant of adaptive lymphocyte development and function 7,8 , although whether cell-intrinsic metabolic signals influence ILC biology is unknown. The enzyme arginase-1 (Arg1) has been identified as a marker of fetal intestinal ILC precursors and adult lung ILC2s 9,10 , although the functional significance of Arg1 enzymatic activity in ILCs remains unclear. Arg1 metabolizes the amino acid l-arginine to generate urea and ornithine, which is further metabolized into the downstream metabolites proline and polyamines that drive collagen synthesis and bioenergetic pathways critical for cell proliferation, respectively 11–13 . Although homeostatic l-arginine metabolism occurs primarily in the liver to complete the urea cycle, immune cells can serve as critical extrahepatic sites of Arg1 activity during infection and tissue inflammation 12–16 . Particularly in the context of cancer or type 2 cytokine–driven inflammation in the intestine, liver and skin, Arg1 activity is a key signature of alternatively activated macrophages 15,16 (AAMacs). AAMac-derived Arg1 acts primarily extrinsically, pro- moting wound healing and tissue fibrosis by triggering collagen synthesis by fibroblasts or by limiting T cell responses via nutrient deprivation of l-arginine 14,17–19 . In contrast, evidence supporting the functional significance of AAMac-derived Arg1 enzymatic activ- ity in the lung remains controversial. For example, models target- ing macrophage-specific Arg1 have failed to recapitulate the effects observed in studies using global inhibition of Arg1 to dampen air- way inflammation 19–23 , which suggests that other cell populations might be responsible for Arg1’s ability to promote the development of lung disease. We demonstrate here that Arg1 has a critical cell- intrinsic role in regulating ILC2 metabolism and the development of type 2 inflammation. RESULTS Constitutive Arg1 expression in precursor and mature ILC2s Arg1 expression has been reported in a population of ILC precursors in the fetal intestine and in mature ILC2s in the lung 9,10 . Whether Arg1 is differentially expressed in distinct adult ILC precursors and 1 Jill Roberts Institute for Research in Inflammatory Bowel Disease, Joan and Sanford I. Weill Department of Medicine, Department of Microbiology and Immunology, Weill Cornell Medicine, Cornell University, New York, New York, USA. 2 Department of Immunometabolism, Max Plank Institute of Immunobiology and Epigenetics, Freiburg, Germany. 3 Division of Cellular Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany. 4 Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center, New York, New York, USA. 5 Division of Cardiovascular Surgery, Center for Translational Lung Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. 6 Present addresses: Biogen, Inc., Cambridge, Massachusetts, USA (S.A.S.); Baker Institute for Animal Health, Cornell University College of Veterinary Medicine, Ithaca, New York, USA (E.D.T.W.); Department of Microbiology and Immunology, Cornell University College of Veterinary Medicine, Ithaca, New York, USA (E.D.T.W.); Department of Microbiology & Immunology, University of British Columbia, Vancouver, British Columbia, Canada (L.C.O.); Faculty of Life Sciences, The University of Manchester, Manchester, UK (M.R.H.). Correspondence should be addressed to D.A. ([email protected]). Received 21 December 2015; accepted 24 February 2016; published online 4 April 2016; doi:10.1038/ni.3421 Arginase 1 is an innate lymphoid-cell-intrinsic metabolic checkpoint controlling type 2 inflammation Laurel A Monticelli 1 , Michael D Buck 2 , Anne-Laure Flamar 1 , Steven A Saenz 1,6 , Elia D Tait Wojno 1,6 , Naomi A Yudanin 1 , Lisa C Osborne 1,6 , Matthew R Hepworth 1,6 , Sara V Tran 1 , Hans-Reimer Rodewald 3 , Hardik Shah 4 , Justin R Cross 4 , Joshua M Diamond 5 , Edward Cantu 5 , Jason D Christie 5 , Erika L Pearce 2 & David Artis 1 Group 2 innate lymphoid cells (ILC2s) regulate tissue inflammation and repair after activation by cell-extrinsic factors such as host-derived cytokines. However, the cell-intrinsic metabolic pathways that control ILC2 function are undefined. Here we demonstrate that expression of the enzyme arginase-1 (Arg1) during acute or chronic lung inflammation is a conserved trait of mouse and human ILC2s. Deletion of mouse ILC-intrinsic Arg1 abrogated type 2 lung inflammation by restraining ILC2 proliferation and dampening cytokine production. Mechanistically, inhibition of Arg1 enzymatic activity disrupted multiple components of ILC2 metabolic programming by altering arginine catabolism, impairing polyamine biosynthesis and reducing aerobic glycolysis. These data identify Arg1 as a key regulator of ILC2 bioenergetics that controls proliferative capacity and proinflammatory functions promoting type 2 inflammation. ARTICLES npg © 2016 Nature America, Inc. All rights reserved.
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656 VOLUME 17 NUMBER 6 JUNE 2016 nature immunology
The innate lymphoid cell (ILC) family orchestrates immunity, inflammation, metabolic homeostasis and tissue repair in multiple tissues, including intestine, liver, adipose, skin and lung1,2. Group 2 ILCs (ILC2s) are the dominant ILC population in human and murine lung, where they act as key initiators of allergen- and non-allergen-induced type 2 inflammation1,3–5 and promote airway tissue repair6. ILC2s are activated by cell-extrinsic environmental cues such as the cytokines interleukin-25 (IL-25), IL-33 and thymic stromal lymphopioetin1,2. However, the cell-intrinsic pathways that regulate ILC2 effector function remain poorly characterized. In particular, regulation of cell metabolism is a critical determi-nant of adaptive lymphocyte development and function7,8, although whether cell-intrinsic metabolic signals influence ILC biology is unknown. The enzyme arginase-1 (Arg1) has been identified as a marker of fetal intestinal ILC precursors and adult lung ILC2s9,10, although the functional significance of Arg1 enzymatic activity in ILCs remains unclear.
Arg1 metabolizes the amino acid l-arginine to generate urea and ornithine, which is further metabolized into the downstream metabolites proline and polyamines that drive collagen synthesis and bioenergetic pathways critical for cell proliferation, respectively11–13. Although homeostatic l-arginine metabolism occurs primarily in the liver to complete the urea cycle, immune cells can serve as critical
extrahepatic sites of Arg1 activity during infection and tissue inflammation12–16. Particularly in the context of cancer or type 2 cytokine–driven inflammation in the intestine, liver and skin, Arg1 activity is a key signature of alternatively activated macrophages15,16 (AAMacs). AAMac-derived Arg1 acts primarily extrinsically, pro-moting wound healing and tissue fibrosis by triggering collagen synthesis by fibroblasts or by limiting T cell responses via nutrient deprivation of l-arginine14,17–19. In contrast, evidence supporting the functional significance of AAMac-derived Arg1 enzymatic activ-ity in the lung remains controversial. For example, models target-ing macrophage-specific Arg1 have failed to recapitulate the effects observed in studies using global inhibition of Arg1 to dampen air-way inflammation19–23, which suggests that other cell populations might be responsible for Arg1’s ability to promote the development of lung disease. We demonstrate here that Arg1 has a critical cell-intrinsic role in regulating ILC2 metabolism and the development of type 2 inflammation.
RESULTSConstitutive Arg1 expression in precursor and mature ILC2sArg1 expression has been reported in a population of ILC precursors in the fetal intestine and in mature ILC2s in the lung9,10. Whether Arg1 is differentially expressed in distinct adult ILC precursors and
1Jill Roberts Institute for Research in Inflammatory Bowel Disease, Joan and Sanford I. Weill Department of Medicine, Department of Microbiology and Immunology, Weill Cornell Medicine, Cornell University, New York, New York, USA. 2Department of Immunometabolism, Max Plank Institute of Immunobiology and Epigenetics, Freiburg, Germany. 3Division of Cellular Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany. 4Donald B. and Catherine C. Marron Cancer Metabolism Center, Memorial Sloan Kettering Cancer Center, New York, New York, USA. 5Division of Cardiovascular Surgery, Center for Translational Lung Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA. 6Present addresses: Biogen, Inc., Cambridge, Massachusetts, USA (S.A.S.); Baker Institute for Animal Health, Cornell University College of Veterinary Medicine, Ithaca, New York, USA (E.D.T.W.); Department of Microbiology and Immunology, Cornell University College of Veterinary Medicine, Ithaca, New York, USA (E.D.T.W.); Department of Microbiology & Immunology, University of British Columbia, Vancouver, British Columbia, Canada (L.C.O.); Faculty of Life Sciences, The University of Manchester, Manchester, UK (M.R.H.). Correspondence should be addressed to D.A. ([email protected]).
Received 21 December 2015; accepted 24 February 2016; published online 4 April 2016; doi:10.1038/ni.3421
Arginase 1 is an innate lymphoid-cell-intrinsic metabolic checkpoint controlling type 2 inflammationLaurel A Monticelli1, Michael D Buck2, Anne-Laure Flamar1, Steven A Saenz1,6, Elia D Tait Wojno1,6, Naomi A Yudanin1, Lisa C Osborne1,6, Matthew R Hepworth1,6, Sara V Tran1, Hans-Reimer Rodewald3, Hardik Shah4, Justin R Cross4, Joshua M Diamond5, Edward Cantu5, Jason D Christie5, Erika L Pearce2 & David Artis1
Group 2 innate lymphoid cells (ILC2s) regulate tissue inflammation and repair after activation by cell-extrinsic factors such as host-derived cytokines. However, the cell-intrinsic metabolic pathways that control ILC2 function are undefined. Here we demonstrate that expression of the enzyme arginase-1 (Arg1) during acute or chronic lung inflammation is a conserved trait of mouse and human ILC2s. Deletion of mouse ILC-intrinsic Arg1 abrogated type 2 lung inflammation by restraining ILC2 proliferation and dampening cytokine production. Mechanistically, inhibition of Arg1 enzymatic activity disrupted multiple components of ILC2 metabolic programming by altering arginine catabolism, impairing polyamine biosynthesis and reducing aerobic glycolysis. These data identify Arg1 as a key regulator of ILC2 bioenergetics that controls proliferative capacity and proinflammatory functions promoting type 2 inflammation.
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mature ILC populations, as well as how this might be influenced by the tissue microenvironment, remains poorly characterized. Examination of bone marrow hematopoietic stem cells (HSCs), common lymphoid progenitors (CLPs) and the common innate lymphoid progenitor (CHILP) in reporter mice that express yellow fluorescent protein under control of the Arg1 promoter (Arg1YFP) did not show any expression of Arg1-YFP (Fig. 1a), which suggests that upregulation of Arg1 in adult bone marrow occurs at a later developmental checkpoint during subset differentiation of the ILC lineages. Consistent with this, analysis of Arg1-YFP expression in lineage-specified bone marrow ILC2 precursors that lack KLRG1 (termed ILC2Ps)24 showed high expression of Arg1-YFP equivalent to that observed in mature KLRG1+ ILC2s (Fig. 1b). Taken together, these data identify Arg1 as a marker of lineage-committed ILC2 pre-cursors in adult bone marrow.
To investigate whether Arg1 expression in mature ILC2s is influ-enced by the cells’ distinct tissue microenvironment, we examined Arg1 expression in ILC2s isolated from various lymphoid, mucosal and adipose tissues. ILC2s in the lung expressed high amounts of Arg1, as measured both by intracellular staining and by expression of Arg1-YFP (Fig. 1c). Further flow cytometric profiling of mature KLRG1+ ILC2s isolated from multiple tissues of Arg1YFP mice, includ-ing spleen, mesenteric lymph node, small intestine, large intestine and epididymal white adipose tissue (Fig. 1d), indicated that constitutive
Arg1 expression represents a conserved trait of mature ILC2s across lymphoid and nonlymphoid tissues.
Mouse ILC2s express Arg1 in inflamed lung tissueIn contrast to lymphoid tissues and barrier surfaces such as the intestine that contain all three subsets of ILCs, the murine lung is dominated by ILC2s3,6. Given the role of Arg1 in regulating lung homeostasis in the context of infection and inflammation15,16,25,26, we sought to examine which cell populations are the dominant sources of Arg1. Unbiased flow cytometric analysis of total numbers of Arg1-YFP-expressing cells in lungs of naive Arg1YFP mice showed that the majority of Arg1 was expressed by lineage-negative (Lin−) cells that expressed CD90, CD25, CD127 and IL-33R, identifying them as ILC2s (Fig. 1e,f). Comprehensive examination of several innate and adaptive immune cell lineages from the lungs of Arg1YFP mice confirmed that ILC2s were the dominant cel-lular source of Arg1 under homeostatic conditions (Fig. 1g).
ILC2s can promote type 2 inflammation in the lung in response to a variety of allergic and nonallergic stimuli1,2, although the cell-intrinsic factors regulating ILC2s’ proinflammatory functions remain poorly characterized. To investigate whether ILC2-intrinsic Arg1 influ-ences lung inflammation, we used a mouse model of acute allergen-induced airway inflammation. Intranasal administration of papain to Arg1YFP mice resulted in elevated Arg1 mRNA expression in the lung compared to that in PBS-treated controls (Fig. 2a), correlating
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Figure 1 Constitutive Arg1 expression is a conserved trait of precursor and mature ILC2s across diverse tissue sites. (a) Flow cytometric analysis of Arg1-YFP expression in bone marrow HSCs (Lin−CD90−CD127−c- KithiSca-1hi), CLPs (Lin−CD90−CD127+CD25−α4β7
− Flt3+Sca-1loc-Kitlo) and CHILPs (Lin−CD90−CD127+α4β7
+Flt3−) from Arg1YFP mice. (b) Flow cytometric analysis of Arg1-YFP expression in immature ILC2Ps (Lin−CD127+CD90+IL-33R+KLRG1−) and mature ILC2s (Lin−CD127+CD90+IL-33R+KLRG1+). (c) Flow cytometric analysis of intracellular Arg1 expression or Arg1-YFP expression in mature ILC2s (Lin−CD45+CD90+CD25+IL-33R+) from naive lung tissue of C57BL/6J wild-type mice or Arg1YFP mice. (d) Flow cytometric analysis of Arg1-YFP expression in mature ILC2s (Lin−CD45+CD90+CD127+IL-33R+KLRG1+) from spleen (SPL), mesenteric lymph node (mLN), small intestine lamina propria (SI), large intestine lamina propria (LI) and epididymal white adipose tissue (EWAT) from naive Arg1YFP or wild-type mice. (e) Sequential flow cytometric gating in naive lung tissue from Arg1YFP mice. Lineage-negative gating (Lin) includes markers for CD3, CD5, FcεR1, NK1.1, CD11b, B220 and CD11c. Numbers indicate the frequency of flow cytometric events. (f) Quantification of the relative frequency of Arg1-YFP+ ILC2s (solid circles) versus that of all other cell populations (non-ILC2) (open circles) in naive lung tissue of Arg1YFP mice. *P < 0.001, unpaired Student’s t-test. (g) Representative flow cytometry plots of Arg1-YFP expression in ILC2s (Lin−CD45+CD90+CD25+IL-33R+), alveolar macrophages (CD11b+CD11c+F4/80+Ly6G−CD3−CD19−), CD11b+ dendritic cells (DCs) (CD103−CD11b+CD11c+F4/80−MHCII+), CD103+ DCs (CD103+CD11b−CD11c+F4/80−MHCII+), eosinophils (CD11b+Siglec-F+CD11c−), NK cells (CD3−NK1.1+), CD4+ T cells (CD3+CD4+) and B cells (CD3−CD19+). WT, wild-type. All data are representative of three independent experiments with similar results. N = 3 mice per group except in e,f, for which n = 8 mice. Data shown represent flow cytometric events.
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Figure 2 ILC2s are a main source of Arg1 in the lung during type 2 inflammation. (a) Arg1 mRNA expression in lung tissue of wild-type mice treated with 30 µg of papain or PBS intranasally (i.n.) for 5 d and analyzed on day 6. Values shown were determined by RT-PCR and are relative to those for PBS-treated control mice. (b–g) Analysis results for wild-type and Arg1YFP mice treated with 30 µg of papain or PBS (i.n.) for 5 d and analyzed on day 6. (b) Representative flow cytometry plots of lung ILC2 frequencies, pregated on Lin−CD45+ cells. (c) Arg1-YFP expression in lung ILC2s from Arg1YFP mice. (d,e) Frequency (d) and number (e) of Arg1-YFP+ ILC2s at day 6. (f) Sequential flow cytometric gating of Arg1-YFP expression in lung tissue from papain-treated Arg1YFP mice. Lineage-negative gating (Lin) includes markers for CD3, CD5, NK1.1, CD11b, B220 and CD11c. Numbers indicate the frequency of flow cytometric events. (g) Quantification of the relative frequency of Arg1-YFP+ ILC2s versus that of non-ILC2 populations in lung tissue of papain-treated Arg1YFP mice. All data are representative of three independent experiments with similar results. N = 3 mice per group (a–e) or 5 mice per group (f,g). Data shown are the mean ± s.e.m. *P < 0.001 as determined by unpaired Student’s t-test.
with increased frequencies of ILC2s in the lung parenchyma (Fig. 2b). The ILC2s continued to express high amounts of Arg1-YFP after aller-gen exposure (Fig. 2c), resulting in elevated frequencies and total numbers of Arg1-expressing ILC2s compared to those in PBS-treated control mice (Fig. 2d,e). Further, unbiased analysis of total Arg1-YFP+ cells showed that ILC2s constituted a major source of Arg1 expression in the inflamed lung (Fig. 2f,g). These data suggest that ILC-intrinsic expression of Arg1 influences the development and/or progression of lung inflammation.
Human ILC2s express Arg1 during lung diseaseElevated expression of Arg1 and dysregulation of arginine metabolism have been reported in people diagnosed with asthma25–28, chronic obstructive pulmonary disease29,30 (COPD) or idiopathic pulmonary fibrosis17 (IPF). However, the cellular sources of the enzyme in human lung disease are incompletely understood and presumed to be limited to the myeloid lineage11,12,31. Using primary lung tissue obtained from individuals diagnosed with COPD or IPF, we next sought to determine whether Arg1 is expressed by ILCs during inflammatory lung disease in humans. Flow cytometric analysis of Lin−CD127+ ILCs identified all group 1 ILC (ILC1), ILC2 and group 3 ILC (ILC3) populations in human explant tissues (Fig. 3a), although the total ILC frequency
and subset distribution did not seem to differ significantly between disease states (Supplementary Fig. 1a,b). However, examination of intracellular Arg1 expression showed a pattern of differential expres-sion among ILC subsets in which Arg1 expression by IL-33R+ ILC2s was comparable to that by CD14+CD16+ myeloid cells (Fig. 3b,c). Arg1 expression in ILC2s did not differ significantly between people diagnosed with COPD and those diagnosed with IPF (Supplementary Fig. 1c), which suggests that Arg1 expression might be a more general-ized signature of these innate cells during inflammatory conditions and could represent a novel target for clinical therapies modulating arginine metabolism.
Deletion of ILC-intrinsic Arg1 dampens airway inflammationTo investigate the role of ILC2-derived Arg1, we generated a genetic mouse model that deletes Arg1 from ILCs using a Cre recombinase targeting cells expressing the IL-7Rα chain (Il7rCre/+). Fate-mapping analysis using Il7rCre/+ mice crossed to Rosa26floxSTOP-eYFP mice showed that all lung ILC2s were marked with a history of IL-7Rα expression (Fig. 4a). Notably, although CD4+ T cells, B cells and NK cells were also marked by a history of IL-7Rα expres-sion (Supplementary Fig. 2), none of those cells expressed Arg1-YFP under steady-state conditions or during papain-induced lung
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Figure 3 Human ILC2s express Arg1 during chronic lung disease. (a) Flow cytometric analysis of lung explant tissue from individuals with COPD or IPF identifying Lin−CD127+ ILC2 subsets expressing IL-33R or CRTH2 or neither marker (ILC1 and ILC3). Colored outlines correspond to the key in b; gold denotes Lin– cells. (b) Intracellular Arg1 expression in the indicated cell subsets. (c) Quantification of Arg1 mean fluorescence intensity (MFI) per cell subset. Data in a and b are representative of six COPD and six IPF tissue samples analyzed as part of one experiment. Data in c are the mean ± s.e.m. *P < 0.05, **P < 0.01, two-way analysis of variance (ANOVA). NS, not significant.
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inflammation (Supplementary Fig. 3). Therefore, use of the IL-7Rα Cre recombinase in combination with Arg1flox/flox mice generates a mouse that lacks Arg1 specifically in ILCs (referred to as Arg1∆ILC here). Transcriptional (Fig. 4b) and flow cytometric (Fig. 4c) analysis confirmed efficient deletion of Arg1 expression in lung ILC2s.
To test whether ILC-intrinsic Arg1 has a role in the development of lung inflammation, we treated Arg1flox/flox, Arg1+/+Il7rCre/+ control and Arg1∆ILC mice with PBS or papain and assessed them for immu-nologic and pathologic parameters of type 2 lung inflammation. All groups treated with PBS had equivalent frequencies of lung ILC2s (Fig. 4d,e), which suggests that despite constitutive expression of Arg1, the enzyme was not required for the development, maturation or maintenance of ILC2s. Although exposure to papain resulted in increased frequencies (Fig. 4d,e) and total numbers (Fig. 4f) of ILC2s in the lungs of Arg1flox/flox and Arg1+/+Il7rCre/+ mice compared with PBS-treated controls, loss of ILC-intrinsic Arg1 expression severely diminished papain-induced ILC2 responses in Arg1∆ILC mice (Fig. 4d–f). The lack of an ILC2 response correlated with decreased Arg1 expression in lung homogenates from Arg1∆ILC mice compared to
Arg1flox/flox and Arg1+/+Il7rCre/+ mice (Fig. 4g), consistent with ILC2s being a critical regulator of Arg1 in the inflamed lung.
ILC2s are the primary source of the type 2 cytokines IL-5 and IL-13 during acute papain-induced inflammation, and these cytokines are critical for driving eosinophilia and the type 2 helper T (TH2) cell subset of CD4+ T cell responses, respectively4,5. Correlating with the reduction in ILC2 numbers, expression of Il5 (Fig. 4h) and Il13 (Fig. 4i) in lung homogenates was significantly decreased in papain-treated Arg1∆ILC mice compared to Arg1flox/flox and Arg1+/+
Il7rCre/+ controls. Further, we observed decreased frequencies of IL-5+IL-13+ ILC2s in Arg1∆ILC mice compared to those in Arg1flox/flox mice (Fig. 4j,k), demonstrating that Arg1 expression is critical for optimal ILC2 cytokine effector function.
Although papain administration resulted in elevated numbers of CD45+ immune cells (primarily eosinophils) in the broncho-alveolar lavage fluid compared to those in control mice (Fig. 4l), this was significantly reduced in the absence of ILC-intrinsic Arg1 (Fig. 4l,m). Additionally, histological examination of the lungs showed that although exposure to papain resulted in clusters
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Figure 4 Absence of ILC-intrinsic Arg1 restrains ILC2 responses and dampens airway inflammation. (a) Flow cytometric fate-mapping analysis of IL-7Rα expression on lung ILC2s from naive Il7rCre/+Rosa26floxSTOP-eYFP mice or wild-type mice, pre-gated on Lin−CD45+ cells. (b) Arg1 mRNA expression in sort-purified lung ILC2s (Lin−CD45+CD90+CD127+CD25+IL-33R+) from Arg1flox/flox and Arg1∆ILC mice at day 6 of papain treatment, determined by RT-PCR and normalized to β-actin levels. (c) Representative flow cytometry histogram of intracellular Arg1 expression in naive lung ILC2s (Lin−CD45+CD90+CD25+) from the indicated mice. (d–n) Analysis results for Arg1flox/flox, Arg1+/+Il7rCre/+ and Arg1∆ILC mice treated with 30 µg of papain or PBS i.n. for 5 d and assessed on day 6. (d–f) Representative flow cytometry plots in which numbers indicate the frequency of flow cytometric events (d), total frequencies (e) and cell numbers (f) of lung ILC2s. Key in e applies to e–i,l,m. (g–i) Expression of Arg1 (g), Il5 (h) and Il13 (i) mRNA in lung tissue, determined by RT-PCR and presented relative to levels in PBS-treated Arg1flox/flox mice. (j,k) Representative flow cytometry plots in which numbers indicate flow cytometric events (j) and frequencies (k) of lung ILC2s expressing IL-5 and IL-13. ILC2s gated as live, Lin−CD45+CD90+CD25+IL-33R+. (l,m) Total cell numbers of CD45+ immune cells (l) and eosinophils (CD11b+Siglec-F+CD11c−) (m) in bronchoalveolar lavage fluid (BALF) at day 6 after treatment. (n) Periodic acid–Schiff (PAS) staining of lung sections in PBS- or papain-treated mice. Red arrows denote PAS+ goblet cells in the airways, and yellow arrows denote foci of immune cell infiltration. Scale bars, 100 µm for large-field images (top two rows) or 50 µm for magnified images (bottom row). All data are representative of three independent experiments with similar results. N = 3 (PBS) or 4 (papain) mice per group. Data shown are the mean and s.e.m. *P < 0.001, unpaired Student’s t-test.
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of immune cell infiltrates and severe goblet cell hyperplasia in Arg1flox/flox and Arg1+/+Il7rCre/+ mice relative to PBS-treated controls, deletion of ILC-intrinsic Arg1 effectively blocked the development of airway inflammation (Fig. 4n). Collectively, these results define an essential role for ILC-intrinsic Arg1 expression in promoting acute type 2 inflammation in the lung.
Arg1 regulates ILC2 responses in chronic lung inflammationIn addition to their role in regulating the development of acute lung inflammation, ILC2s have been implicated in the progression or resolution of chronic lung inflammation, including in IPF32 and helminth-parasite-induced lung remodeling33,34. We next sought to test the involvement of ILC-derived Arg1 during chronic lung inflammation. Infection with the helminth parasite Nippostrongylus brasiliensis leads to the development of chronic lung inflammation characterized by increased amounts of type 2 cytokines and height-ened Arg1 expression35,36. Although Arg1flox/flox mice exhibited a twofold increase in the frequency of ILC2s in the inflamed lung 1 month after infection, compared to naive controls, loss of ILC-intrin-sic Arg1 prevented the generation of ILC2 responses (Supplementary Fig. 4a,b) and was associated with decreased Arg1 expression in the inflamed lung (Supplementary Fig. 4c). Similarly, in a mouse model of elastase enzyme instillation that leads to chronic damage to the alveolar septa partially mimicking the emphysematous pathology observed in COPD37,38, we found that absence of ILC-intrinsic Arg1 prevented the generation of robust ILC2 responses (Supplementary Fig. 4d,e), was associated with decreased Arg1 expression in lungs (Supplementary Fig. 4f) and reduced the severity of emphysematous lung pathology (Supplementary Fig. 4g). Taken together, these data highlight a conserved role for ILC-intrinsic Arg1 in controlling ILC2 responses in both acute and chronic models of lung inflammation.
ILC3 function is independent of ILC-intrinsic Arg1Expression of Arg1 is not restricted to the ILC2 lineage, as fetal RORγt+ lymphoid-tissue-inducer (LTi) cells and a proportion of adult intestinal RORγt+ LTi-like ILC3s have been reported to express Arg1-YFP10. Therefore, we next sought to test whether ILC-intrinsic Arg1 can regulate ILC3 responses in the steady state or in the context of bacteria-induced intestinal inflamma-tion. Examination of gut-associated RORγt+ ILC3s and GATA3hi ILC2s in Arg1flox/flox and Arg1∆ILC mice showed equivalent total ILC frequencies (Supplementary Fig. 5a,b) and ILC3/ILC2 ratios (Supplementary Fig. 5c,d), which suggests that deletion of ILC-intrinsic Arg1 does not impair the development or homeosta-sis of ILC3s. To test whether the functionality of ILC3s is affected by the absence of ILC-intrinsic Arg1, we infected mice with the intestinal bacterium Citrobacter rodentium. ILC3-derived IL-22 responses are critical mediators of host protection in C. rodentium infection39. Whereas mice lacking RORγt+ cells (RORc(γt)gfp/gfp) or innate and adaptive lymphocytes (Rag2−/−Il2rg−/− and Rag1−/−) suc-cumbed to infection within 2 weeks after the bacterium was intro-duced, Arg1flox/flox and Arg1∆ILC mice did not show any evidence of Citrobacter-induced inflammation or disease and survived infection (Supplementary Fig. 5e). Collectively, these data demonstrate that Arg1 activity controls optimal ILC2, but not ILC3, responses.
Lung inflammation is independent of myeloid Arg1In addition to its association with ILCs, Arg1 expression is an effector signature of AAMacs, although the functional significance of myeloid-derived Arg1 activity during type 2 inflammation in the lung remains controversial19–23. Fate-mapping analysis of Il7rCre/+ Rosa26floxSTOP-eYFP mice showed that a minor population of macro-phages showed a history of IL-7Rα expression (Fig. 5a), and only a
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Figure 5 Development of airway inflammation is dependent on ILC-intrinsic, not myeloid- cell-intrinsic, expression of Arg1. (a,b) Flow cytometric analysis of alveolar macrophages (CD11b+CD11c+F4/80+Ly6G−CD3−CD19−) in (a) Il7rCre/+Rosa26floxSTOP-eYFP mice and (b) Arg1YFP mice during steady state and papain-induced airway inflammation at day 6 after treatment. (c–e) Analysis results for Arg1flox/flox and Arg1∆ILC mice treated with 30 µg of papain or PBS i.n. for 5 d and assessed on day 6. (c) Total frequencies of lung macrophages (CD11c+CD11b+F4/80+). (d) Arg1 mRNA expression in sort-purified macrophages, normalized to β-actin. (e) Intracellular Arg1 expression in macrophages. FMO, fluorescence minus one. (f–i) Analysis results for Arg1flox/flox, Arg1∆ILC and Arg1∆Lyz2 mice treated with 30 µg of papain or PBS i.n. for 5 d and assessed on day 6. (f,g) Representative flow cytometry plots in which numbers indicate the frequency of flow cytometric events (f) and total frequencies (g) of lung ILC2s (Lin−CD45+CD90+CD25+). (h) Total numbers of eosinophils (CD11b+Siglec-F+CD11c−) in bronchoalveolar lavage fluid. (i) PAS staining of lung sections. Red arrows denote PAS+ goblet cells in the airways, and yellow arrows denote foci of immune cell infiltration. Scale bars, 100 µm for large-field images (top) or 50 µm for magnified images (bottom). All data are representative of two independent experiments with similar results. N = 2 (PBS) or 3 (papain) mice per group. Data shown are the mean and s.e.m. *P < 0.001, unpaired Student’s t-test.
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small proportion of macrophages expressed Arg1-YFP during acute papain-induced inflammation (Fig. 5b). Flow cytometric analysis did not show any significant difference in macrophage frequencies (Fig. 5c), and we observed equivalent expression of macrophage-intrinsic Arg1 mRNA (Fig. 5d) and protein (Fig. 5e) in the inflamed lungs of Arg1flox/flox and Arg1∆ILC mice after papain exposure, indicat-ing that AAMac accumulation and Arg1 expression are not impaired in Arg1∆ILC mice.
To test whether myeloid sources of Arg1 can act extrinsically to influence the magnitude of ILC2 responses and/or the development of papain-induced airway inflammation, we deleted Arg1 expres-sion in macrophages and neutrophils using Cre recombinase under control of the Lyz2 (LysM) promoter19,40 (Arg1∆Lyz2). Absence of Arg1 in LysM+ myeloid cells did not prevent the generation of robust lung ILC2 responses to papain (Fig. 5f,g), nor did it influence the development of airway eosinophilia (Fig. 5h) or lung inflammation (Fig. 5i). Taken together, these results indicate a selective requirement for ILC-intrinsic Arg1 in the development of acute airway inflam-mation, revealing an unexpected degree of cellular specificity with respect to the role of this enzyme in the lung.
Arg1 controls optimal ILC2 proliferationThe lack of a robust ILC2 response in the absence of cell-intrinsic Arg1 metabolic function could be due to impaired cell survival and/or reduced proliferative capacity. We found that deletion of Arg1 had no significant effect on the frequencies of 7AAD+annexin V+ apop-totic ILC2s in the lungs of either PBS-treated or papain-treated mice (Supplementary Fig. 6a–c), which suggests that the absence of ILC-intrinsic Arg1 does not influence ILC2 survival under homeostatic conditions or during acute lung inflammation. To test whether loss of Arg1 affects ILC2 proliferation, we examined intracellular expres-sion of the proliferation marker Ki67 in ILC2s isolated ex vivo dur-ing acute papain-induced lung inflammation. A high proportion of lung ILC2s from Arg1flox/flox and Arg1+/+Il7rCre/+ mice were Ki67+ cells (38–44%) (Fig. 6a,b), indicating that those mice mounted a strong effector response to the papain allergen. In contrast, deletion of ILC-intrinsic Arg1 severely restrained ILC2 proliferation, result-ing in significantly decreased frequencies (Fig. 6a,b) and cell num-bers (Fig. 6c) of Ki67+ ILC2s compared to Arg1flox/flox and Arg1+/+ Il7rCre/+ mice. To test directly whether cell-intrinsic Arg1 enzymatic activity is required for regulation of ILC2 proliferation, we sort- purified activated ILC2s from wild-type mice treated with recombinant murine IL-33 (rmIL-33) and tracked cellular division in the presence
or absence of the selective Arg1 inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) (Fig. 6d). Within 48 h, nearly 80% of the ILC2s treated with dimethylsulfoxide (DMSO; controls) had undergone at least one division, and more than 20% had undergone three to five divisions (Fig. 6e,f). Compared to controls, cells subjected to Arg1 enzymatic inhibition showed greatly decreased division, with more than 45% of the cells remaining undivided (Fig. 6e,f). Consistent with our in vivo papain studies, the lack of division by the nor-NOHA-treated cells was not associated with increased cell death (Supplementary Fig. 6d). Collectively, these studies demonstrate an essential role for Arg1 enzymatic activity in controlling the maximal proliferative capacity, but not survival, of activated ILC2s.
Arg1 inhibition disrupts amino acid metabolismArginine can be catabolized through several enzymatic pathways to yield a diverse set of metabolic intermediates that serve as substrates for downstream cellular and bioenergetic processes (Supplementary Fig. 7a and refs. 11,31,41,42). We hypothesized that the lack of proliferation in Arg1-deficient ILC2s might be associated with an imbalance among these metabolic substrates due to the disruption of Arg1 activity and possible compensation by the other enzymatic pathways to catabolize the excess arginine. To examine this, we first cultured IL-33-activated ILC2s in the presence or absence of nor-NOHA and measured the relative abundance of metabolites using liquid chromatography coupled with mass spectrometry (Supplementary Fig. 7b). As expected, inhibition of Arg1 resulted in increased amounts of arginine in nor-NOHA-treated cells (Fig. 7a), confirming that ILC2s express functional Arg1. Whereas DMSO-treated cells showed high abundance of the immediate downstream products ornithine and proline, the amounts of these metabolites were comparatively severely diminished in cells subjected to Arg1 inhibition (Fig. 7b,c). We did not observe any corresponding increase in metabolites associated with two other main arginine catabolic pathways that would generate either citrulline-argininosuccinate via nitric oxide synthase (Fig. 7d) or creatine-creatinine biosynthesis via l-arginine:glycine amidinotransferase (Fig. 7e), which suggested that excess arginine is probably not diverted through these pathways to compensate for the absence of Arg1.
Figure 6 Cell-intrinsic Arg1 controls optimal ILC2 proliferation. (a–c) Analysis results for Arg1flox/flox, Arg1+/+Il7rCre/+ and Arg1∆ILC mice treated with 30 µg of papain or PBS i.n. for 5 d and assessed on day 6 for lung ILC2 proliferation by Ki67 staining. Shown are representative flow cytometry plots in which numbers indicate the frequency of flow cytometric events (a), Ki67+ ILC2 frequencies (Lin−CD45+CD90+CD25+) (b) and total cell numbers (c). (d–f) Analysis results for ILC2s sort- purified from rmIL-33-treated wild-type mice and then cultured in vitro in the presence or absence of the Arg1 inhibitor nor-NOHA. Shown are a representative histogram (d) and the ILC2 percentage in each cell division (e,f) for sort-purified lung ILC2s (Lin−CD45+CD90+CD127+CD25+IL-33R+) labeled with Cell Trace Violet, cultured for 48 h (with rmIL-2, rmIL-7, rmIL-33 in DMSO or nor- NOHA) and assessed for dilution of cell-trace dye by flow cytometry. All data are representative of three independent experiments with similar results. N = 3 mice per group. Data shown are the mean and s.e.m. *P < 0.01, **P < 0.001, unpaired Student’s t-test.
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Changes in the size of a metabolite pool can result from either altered production or an altered consumption rate within the cell41,43. Therefore, to further examine the metabolic fate of arginine in acti-vated ILC2s, we traced the incorporation of uniformly enriched U-13C-l-arginine isotope in ILC2s in the presence or absence of Arg1 activity. Whereas we observed rapid and almost complete entry of 13C-l-arginine into ornithine (Fig. 7f,g) and proline (Fig. 7h,i) in DMSO-treated ILC2s, with saturation kinetics being quickly achieved, this process was severely diminished in cells treated with Arg1 inhibitor, indicating that arginine is the primary precursor for both molecules in activated ILC2s. Further, inhibi-tion of Arg1 activity also resulted in severely diminished entry of 13C-l-arginine into biosynthesis of the ornithine-derived polyamine metabolite spermidine (Fig. 7j,k). Notably, reversible interconversion between proline and ornithine metabolite pools is a well-appreciated reaction in mammalian cells42, supporting the conclusion that ornithine-derived polyamines, rather than ornithine and proline themselves, are a primary fate of l-arginine catabolism in ILC2s.
Taken together, these data indicate a model in which disruption of Arg1 in ILC2s is not associated with enhanced metabolism of nitric oxide synthase or l-arginine:glycine amidinotransferase (Supplementary Fig. 7a) but does prevent the generation of l-arginine-derived polyamines (Supplementary Fig. 7a), molecules that are known to regulate cell growth and survival31,41.
Arg1 regulates maximal ILC2 glycolytic capacityProteins are just one component of a diverse set of nutrient substrates that immune cells can use to fuel their growth7,44, raising the ques-tion of whether Arg1 metabolism influences bioenergetic pathways in addition to amino acid catabolism. In particular, the induction of aerobic glycolysis is a hallmark of effector lymphocyte metabo-lism7, although whether this reprogramming is used by ILCs during activation has never been examined. First, to characterize the bioenergetic profile of ILC2s during activation, we sort-purified lung IL-33R+ ILC2s and TH2 cells from mice treated with rmIL-33 (Fig. 8a) and measured the cellular oxygen-consumption rate (OCR) and extracellular acidification rate (ECAR), indicators of mitochondrial respiration and aerobic glycolysis, respectively. ILC2s showed both elevated spare respiratory capacity (Fig. 8b,c) and high ECAR (Fig. 8d,e) in comparison to TH2 cells, suggesting that ILC2s possess the capacity to augment mitochondrial respiration above basal conditions compared to TH2 cells and strongly engage aero-bic glycolysis. To test specifically whether Arg1 enzymatic activity
can influence the respiratory or glycolytic capacity of ILC2s, we activated lung ILC2s with rmIL-33 in vivo and treated the cells ex vivo with nor-NOHA to inhibit Arg1 activity during measurement of ECAR and OCR. ILC2 spare respiratory capacity was not sub-stantially affected by Arg1 inhibition (Fig. 8f,g), which suggests that this enzyme does not have a notable effect on oxidative phosphor-ylation in ILC2s. In contrast, inhibition of Arg1 resulted in severely diminished maximal glycolytic capacity (Fig. 8h,i), which indicates a critical regulatory role for Arg1 in influencing glucose metabolism in activated ILC2s.
Together with the trace analysis of arginine metabolites, these data provide evidence for a previously unrecognized aspect of this bio-chemical pathway in which Arg1 enzymatic activity acts on multiple biosynthetic and bioenergetic pathways to function as a metabolic checkpoint that controls ILC2 proliferation and effector function driving type 2 inflammation.
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Figure 7 Inhibition of cell-intrinsic Arg1 disrupts the balance of amino acid metabolites and impairs polyamine synthesis in ILC2s. (a–e) Analysis of sort-purified lung ILC2s from rmIL-33-treated wild-type mice cultured with rmIL-2, rmIL-7 and rmIL-33 in DMSO or nor-NOHA for 24 h. Results of liquid chromatography–mass spectrometry analysis for abundance of (a) arginine, (b) ornithine, (c) proline, (d) citrulline and (e) creatine. (f–k) Analysis of sort-purified lung ILC2s from rmIL-33-treated wild-type mice cultured with U-13C-l-arginine (13C L-arg) with rmIL-2, rmIL-7, rmIL-33 and DMSO or nor-NOHA for 18 h. Incorporation kinetics and relative abundance of the U-13C-l-arginine isotope tracer into (f,g) ornithine, (h,i) proline and (j,k) spermidine, as determined by liquid chromatography–mass spectrometry isotope-tracing analysis. Data are representative of two independent experiments with similar results. N = 3 biological replicates per condition. (f,h,j) Data represent individual biological replicates (circles) and means (curves) where N = 3 biological replicates. Data shown are the mean and s.d. for a–e,g,i,k. *P < 0.05, **P < 0.001, unpaired Student’s t-test (a–c) or two-way ANOVA with Bonferroni post-test correction (f,h,j).
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DISCUSSIONILC2s can promote pathologic inflammation or host-protective tissue repair in the lung after activation by cell-extrinsic environmental cues such as alarmins and cytokines1,2. However, the cell-intrinsic meta-bolic pathways that regulate ILC2 effector function are undefined. Here we demonstrate a selective and essential role for cell-intrinsic Arg1 expression in regulating the proliferation and effector function of ILC2s required to promote acute and chronic type 2 airway inflam-mation. Inhibition of Arg1 enzymatic activity disrupted multiple components of ILC2 metabolic programming by altering arginine catabolism and limiting aerobic glycolysis, which indicates that Arg1 enzymatic activity serves as an essential checkpoint controlling ILC2 metabolism and the development of type 2 inflammation.
We found that unlike Arg1 in macrophages, which requires induc-tion by type 2 cytokines15,16, Arg1 was constitutively expressed in bone marrow ILC2 progenitors and maintained in mature cells across diverse tissue environments. This leads to the question of when Arg1 is turned on during ILC development. Although HSCs, CLPs and CHILP did not express Arg1, examination of an ILC2 lineage- committed bone marrow precursor lacking KLRG1 (ILC2P)24 showed Arg1 expression equivalent to that in mature ILC2s. This upregulation of Arg1 expression in ILC2Ps occurs before the cells express transcripts for the effector cytokines IL-5 and IL-13 (ref. 24), which might provide a rationale for why a previous study using IL-5- promoter-driven Cre to delete Arg1 showed no significant effects on ILC2 responses9. Of note, the constitutive Arg1 expression in ILC2s is independent of IL-4– or IL-13–STAT6 signaling9, leaving the molecular and cytokine cues that induce Arg1 expression in need of further investigation.
Until the identification of ILC2s as a constitutive source of Arg1, expression of this enzyme was thought to be a defining trait of the myeloid lineage, particularly of AAMacs, in which it serves to regulate wound healing, fibrosis and suppression of T cell responses11,14–16. Our study suggests an unexpected degree of cellular specificity in the role of this enzyme, as deletion of myeloid cell sources of Arg1 via Lyz2-promoter-driven expression of Cre recombinase did not
affect ILC2 responses or dampen papain-induced airway inflamma-tion. Although further analysis using additional models of myeloid cell depletion is required to determine the reasons for this cellular compartmentalization of Arg1 function, it may provide a rationale for reconciling paradoxical reports of the role of Arg1 during type 2 lung inflammation. Previous studies using deletion of myeloid-cell-intrinsic Arg1 failed to recapitulate the effects observed when global chemical inhibition or absence of hematopoietic-cell-restricted Arg1 dampens airway inflammation19–23. The data presented here demon-strate that deletion of ILC-intrinsic Arg1 acts to limit the magnitude of the ILC2 response itself, thereby diminishing the cells’ proinflam-matory capacity and preventing the development of acute and chronic type 2 inflammation in the lung. Notably, myeloid-cell-intrinsic Arg1 expression must be induced via AAMac polarization, a process that is partially dependent on ILC2-derived type 2 cytokines45,46, thus revealing an additional, indirect mechanism by which ILC2s control Arg1 metabolism during inflammatory conditions.
Elevated ILC2 responses have been reported in a number of human inflammatory disorders of the intestine, skin and lung barrier sites1. Particularly in the lung, dysregulation of L-arginine metabolism and Arg1 expression has been observed in people diagnosed with inflam-matory diseases such as asthma25–28, COPD29,30 and IPF17. Further, dietary supplementation with l-arginine and chemical inhibition of Arg activity are being evaluated as clinical therapies for the treatment of asthma25,26,28. Despite these clinical correlations, the mechanisms by which this enzyme regulates lung inflammation are incompletely understood and are assumed to be largely due to a reciprocal effect of increased nitric oxide and smooth muscle contractility in the absence of Arg1 (refs. 26,30). Further, the identities of the sources of human Arg1 are controversial and incompletely defined. Using primary lung tissue from people with COPD and IPF, we observed selective expres-sion of Arg1 by IL-33R+ ILC2 populations but not by other ILC subsets. Although the significance of this restricted expression pattern is not yet clear, IL-33R expression is strongly enhanced on human ILC2s in response to cytokine signals47 and therefore may represent a more activated state that ILC2s gain in inflamed tissue. Taken together with
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DMSO nor-NOHAe f g h iFigure 8 Arg1 controls the maximal glycolytic capacity of activated ILC2s. (a–e) Analysis of sort-purified lung ILC2s and TH2 cells from rmIL-33- treated Foxp3eGFP reporter mice. Shown are results for (a) flow cytometric gating where numbers represent the frequency of flow cytometric events, (b) OCR, (c) spare respiratory capacity (SRC; % max OCR, of baseline OCR, after injection with the mitochondrial uncoupler FCCP), (d) ECAR and (e) maximal glycolytic capacity (% max ECAR of baseline ECAR) as determined by Seahorse metabolic profiling in response to oligomycin (Oligo), FCCP, and rotenone plus antimycin A (R/A). (f–i) Analysis of sort-purified ILC2s from rmIL-33-treated wild-type mice by Seahorse metabolic profiling with DMSO or nor-NOHA injected during measurement. Shown are results for (f) OCR, (g) SRC, (h) ECAR and (i) maximal glycolytic capacity. Data in a–e are combined from two experiments; n = 3 TH2 cell replicates and 12 ILC2 replicates. Data in f and h are from one representative experiment out of four experiments with similar results; n = 7 DMSO replicates and 7 nor-NOHA replicates. Data in g and i are combined from four experiments; n = 18 DMSO replicates and 16 nor-NOHA replicates. Data in b–i are the mean ± s.e.m. Individual circles in b,d,f,h represent replicate cell samples analyzed by the Seahorse Analyzer. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired Student’s t-test.
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the mouse mechanistic data, our studies suggest that Arg1 metabolic control of ILC2 function is a critical determinant of both acute and chronic type 2 inflammation in the lung and thus may represent a pre-viously unrecognized axis for improvement of targeted therapeutics.
Despite constitutive expression of Arg1 in bone marrow ILC2 pro-genitors, the absence of Arg1 did not seem to affect ILC2 develop-ment or maturation. Instead, Arg1 activity profoundly influenced the cells’ ability to proliferate and exert their proinflammatory function after activation. This suggests a clear demarcation in the role of this enzyme in regulating the inflammatory, but not the homeostatic, state of ILC2s, perhaps because of the differential metabolic requirements of proliferating cells compared to those at rest. Inhibition of Arg1 and the subsequent decreased catabolism of arginine into ornithine resulted in an imbalance of amino acid metabolites, especially reduced synthesis of polyamines from l-arginine. Polyamine metabolites have been linked to diverse biological processes such as proliferation, apoptosis and cellular stress responses31,41, and further biochemical studies will be required to investigate the fates of these molecules in ILC2 biology.
Amino acids are one of a large array of nutrients used for cell growth7,44, provoking the question of whether Arg1 influences other aspects of ILC2 metabolic programming. We found that the effect of Arg1 was not limited to arginine catabolism—it also resulted in severe inhibition of the ability of these cells to engage in maximal rates of aerobic glycolysis (also known as Warburg metabolism7,8) to fuel proliferation. Although the effects of Arg1 on arginine and glu-cose metabolism might not be directly linked and probably involve a complex imbalance of many metabolic intermediates, it is remarkable that disruption of a single urea-cycle enzyme profoundly influences such distinct metabolic pathways, and this may lead to a new under-standing of the importance of this enzyme beyond the classical urea cycle. As both a constitutive and an inflammatory source of arginase, ILC2s seem to occupy a unique functional niche mimicking both Arg1-dependent homeostatic hepatocyte function and inflammation- induced myeloid cell function. Critically, however, mammalian sur-vival depends on hepatocyte-intrinsic Arg1 expression above any hematopoietic source of the enzyme12,31, perhaps reflecting an evo-lutionary compartmentalization of arginase activity that fulfills the primary biological need of mammals for urea production while also limiting the proinflammatory consequences of Arg1-driven ILC2 activation. Understanding how Arg1 metabolism is integrated into the broader metabolic networks that govern the ability of ILCs and other innate immune cells to meet the biosynthetic and bioenergetic demands created during tissue inflammation may reveal new thera-peutic targets for the treatment of numerous human inflammatory and metabolic diseases.
METHODSMethods and any associated references are available in the online version of the paper.
Note: Any Supplementary Information and Source Data files are available in the online version of the paper.
ACkNOWLEDgMENTSWe thank members of the D. Artis and G.F. Sonnenberg laboratories for critical reading of this manuscript. We thank R.M. Locksley (University of California, San Francisco, San Francisco, California, USA) for generously providing the Arg1YFP mice and B. Vallance (University of British Columbia, Vancouver, British Columbia, Canada) for providing Citrobacter rodentium. This work was supported by the National Institutes of Health (NIH) (grants AI061570, AI087990, AI074878, AI083480, AI095466, AI095608, AI102942 and AI097333 to D.A.; T32-AI007532
to L.A.M.; CA181125 and AI091965 to E.L.P.; HL087115, HL081619, HL096845, HL115354 and HL114626 to J.D.C.; HL116656 to E.C.; and HL090021 and K23-HL121406 to J.M.D.), the Burroughs Wellcome Fund (D.A. and E.L.P.), the Crohn’s & Colitis Foundation of America (D.A. and M.R.H.), the Edmond J. Safra Foundation/Cancer Research Institute (L.C.O.), the National Science Foundation (grant DGE-1143954 to M.D.B.), the Robert Wood Johnson Foundation (grant AMFDP 70640 to E.C.), the Thoracic Surgery Foundation (E.C.) and the German Research Foundation (DFG SFB 873–project 11 to H.-R.R.). We thank the Mucosal Immunology Studies Team (MIST) of the NIH NIAID for shared expertise and resources.
AUTHOR CONTRIBUTIONSL.A.M. carried out mouse experiments with technical assistance from A.-L.F., N.A.Y., L.C.O., M.R.H. and S.V.T. M.D.B. and E.L.P. carried out Seahorse metabolic assays. H.S. and J.R.C. carried out liquid chromatography–mass spectrometry experiments. J.M.D., E.C. and J.D.C. provided human tissue samples. L.A.M., S.A.S. and E.D.T.W. carried out experiments involving human tissue samples. H.-R.R. provided Il7rCre/+ mice. L.A.M. and D.A. conceived of the study, designed experiments, analyzed data and wrote the manuscript.
COMPETINg FINANCIAL INTERESTSThe authors declare no competing financial interests.
reprints and permissions information is available online at http://www.nature.com/reprints/index.html.
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ONLINE METHODSMice. C57BL/6J (wild-type) mice were purchased from the Jackson Laboratory. Arg1flox/flox, Lyz2-Cre, RORc(γt)gfp/gfp, Foxp3eGFP, Rag1−/− and Rosa26floxSTOP-eYFP mice on a C57BL/6J background were purchased from the Jackson Laboratory and bred in-house. Rag2−/−Il2rg−/− mice were from Taconic Farms (Hudson, NY). Arg1YFP mice on a C57BL/6 background9,10 were generously provided by Richard Locksley (University of California, San Francisco). Il7rCre/+ mice on a C57BL/6 background48 were provided by H.-R.R. and were used only as heterozygotes. For all Cre-flox studies, littermate controls were used and animals were cohoused after weaning. Male and female mice were used at 7–20 weeks of age. In individual experiments, all animals were age- and sex-matched; exact numbers of animals used per experiment are indicated in figure legends. All mice were maintained in specific-pathogen- free facilities at the University of Pennsylvania or Weill Cornell Medicine. All protocols were approved by both the University of Pennsylvania and the Weill Cornell Medicine Institutional Animal Care and Use Committees.
Models of lung and intestinal inflammation. For induction of acute type 2 airway inflammation, we anesthetized mice with Isothesia isoflurane, USP (Henry Schein Animal Health), treated them with 30 µg of papain (Calbiochem) or PBS intranasally (i.n.) in a volume of 30 µl every day for 5 d, and killed them on day 6 for analysis. For induction of helminth-induced chronic type 2 inflammation, we anesthetized mice with Isothesia isoflu-rane, injected them subcutaneously with 500 L3 larvae of Nippostrongylus brasiliensis, and analyzed them 30–32 d after infection. For induction of chronic emphysematous pathology, we anesthetized mice with Isothesia isoflurane, administered 3 units of elastase (Elastin Products Company) or PBS intratracheally, and assessed the mice at 30–35 d after treatment. For induc-tion of intestinal inflammation, we prepared Citrobacter rodentium (formerly Citrobacter freundii; biotype 4280) strain DBS100 (provided by Bruce Vallance, University of British Columbia) by selecting a single colony and culturing it in LB broth overnight as described39. Mice were inoculated with approximately 1 × 1010 colony-forming units in 200 µL via oral gavage.
Isolation of cells from mouse tissue. For isolation of cells from lymphoid tissues, tissues were pressed through 70-µm cell strainers using the plunger of a 3-ml syringe and washed with DMEM wash media (Dulbecco’s modified Eagle’s medium with 10% FBS (Denville Scientific), 1% l-glutamine (Gibco), 1% penicillin–streptomycin (Gibco)), and any remaining red blood cells were lysed. We isolated cells from bone marrow by flushing out the marrow from cleaned bones using a syringe containing DMEM wash media. Red blood cells were lysed with ACK buffer (Lonza). Isolation of cells from bronchoalveolar lavage fluid and lung tissue was done as described6. We flushed the lungs with two washes of 1 ml of cold PBS via a thin tube inserted into a cut made in the trachea. Lungs were then perfused with 10 ml of PBS through the right ven-tricle of the heart before removal. Lung lobes were cut into small pieces with scissors and digested with 2 mg/ml collagenase D (Roche) in PBS for 45 min at 37 °C with vortexing every 10 min. Samples were pressed through 70-µm cell strainers and washed with DMEM wash media, and any remaining red blood cells were lysed with ACK buffer. Isolation of lymphocytes from epididymal white adipose tissue was done as described49. Briefly, tissue was minced with scissors and then digested with 1 mg/ml collagenase type II (Sigma-Aldrich) at 37 °C with shaking at 200 r.p.m. for 60–90 min. Digested tissues were passed through 70-µm cell strainers and washed with DMEM. The adipocyte layer was removed after centrifugation, and red blood cells were lysed with ACK buffer. Isolation of cells from the lamina propria of the large and small intestine was done as described50. Single-cell suspensions from all tissues were used for subsequent flow cytometry staining.
Isolation of cells from human lung tissue. Explanted human lung tissue was collected from patients undergoing lung transplantation who elected to participate in the Prospective Registry of Outcomes in Patients Electing Lung Transplantation (PROPEL) study at the Hospital of the University of Pennsylvania. University of Pennsylvania Institutional Review Board approval and informed written consent from patients were obtained before recruit-ment. We prepared single-cell suspensions of lung by chopping tissue into pieces with scissors, incubating the finely chopped tissue for 1 h with 2 mg/ml
collagenase D (Roche) and 20 µg/ml DNase I (Roche) at 37 °C with shaking at 200 r.p.m., and mashing the digested tissue through a wire mesh sieve. Liquid was passed through 70-µm cell strainers, underlaid with Ficoll-Paque (GE Healthcare), and centrifuged at 1,600 r.p.m. for 20 min at room temperature with the brake off. The white lymphocyte interface layer was removed, and remaining red blood cells were lysed with ACK buffer. Cells were frozen in 90% FBS, 10% DMSO in a Nalgene Mr. Frosty container (Thermo Scientific). For flow cytometric analysis, samples were thawed and stained as a single cohort to ensure consistency.
Flow cytometry. Single-cell suspensions were stained with a combination of fluorescently conjugated monoclonal antibodies. All antibodies were from Affymetrix eBioscience and used at 1:200 dilution unless otherwise specified. For mouse studies, we used antibodies to the following mouse proteins: α4β7 (DATK32), Flt3 (A2F10), KLRG1 (2F1), Ly6G (1A8), F4/80 (BM8), FcεR1 (MAR-1), CD3 (145-2C11), CD4 (GK1.5), CD5 (53-7.3), CD11b (M1/70), CD11c (N418), CD19 (eBio1D3), CD25 (PC61.5), CD45 (30-F11), CD127 (A7R34), CD90.1 (HIS51), CD90.2 (53-2.1), IL-33R (DJ8, MD Bioproducts), NK1.1 (PK136), Siglec-F (E50-2440), CD103 (2E7) and MHCII (M5/114.15.2). Streptavidin (17-4317-82) was also from Affymetrix eBioscience. For mouse studies, lineage-negative gating included markers for CD3, CD5, FcεR1, NK1.1, CD11b, B220 and CD11c. For human studies, we used antibodies to the following human proteins: CD3 (OKT3), CD4 (OKT4), CD5 (UCHT2), CD14 (61D3), CD16 (CB16), CD19 (HIB19), CD56 (B159), CD25 (BC96), CD45 (HI30), CD127 (eBioRDR5; dilution, 1:100), CRTH2 (BM16, BD Biosciences; dilution, 1:50), FcεR1α (AER-37) and IL-33R (ST2L, B4E6, MD Bioproducts; dilution, 1:100). We noted substantial variability in staining efficiency between different lots of fluorescein isothiocyanate (FITC)-conjugated anti-human IL-33R (ST2L, MD Bioproducts) and recommend that the staining efficacy of this antibody be validated for each individual lot.
For measurement of intracellular cytokine expression, cells were isolated ex vivo and stimulated in complete media (DMEM with 10% FBS (Denville Scientific), 1% l-glutamine (Gibco), 1% penicillin–streptomycin (Gibco), 25 mM HEPES buffer, and 55 µM 2-β-mercaptoethanol (Sigma-Aldrich)) with 50 ng/ml phorbol 12-myristate 13-acetate (Sigma-Aldrich), 500 ng/ml ionomycin (Sigma-Aldrich), and 10 µg/ml brefeldin A (Sigma-Aldrich) for 4 h. Cells were subsequently surface-stained with a combination of the antibodies listed above, fixed and permeabilized using a commercially available kit (BD Cytofix/Cytoperm, BD Biosciences), and stained with IL-13–Alexa Fluor 488 (eBioscience) and IL-5–phycoerythrin (eBioscience).
For measurement of intracellular arginase, cells were isolated directly ex vivo, stained with antibodies to surface antigens, fixed and permeabilized according to the manufacturer’s instructions (BD Biosciences Cytofix/CytoPerm) and stained with sheep anti-human or anti-mouse Arg1 conjugated with FITC or phycoerythrin (R&D Systems).
For analysis of transcription factor expression, cells were isolated directly ex vivo, stained with antibodies to surface antigens, fixed and permeabilized according to the manufacturer’s instructions (Foxp3/Transcription Factor Staining Buffer Set, eBioscience) and stained with phycoerythrin-cojugated RORγt (B2D, eBioscience) and PerCP eFluor 710–conjugated GATA3 (TWAJ, eBioscience). Ki67-phycoerythrin (BD Pharmigen), annexin V–FITC (BD Pharmigen) and 7AAD (BD Via-Probe, BD Biosciences) staining was done with commercially available kits (BD Biosciences).
For all stains except those used for annexin V and 7AAD analysis, dead cells were excluded from analysis by means of a viability stain (Live/Dead Fixable Aqua stain, Invitrogen). Samples were acquired on a custom-configuration BD LSRII or BD Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (v9.2, Tree Star).
Cell-trace assays. For analysis of cell division, C57BL/6J mice were treated with 100 ng of rmIL-33 (R&D Systems) i.n. every 3 d for 2–3 weeks. Lin−CD45+CD90+CD25+IL-33R+ ILC2s were sort-purified from lungs and labeled with 5 µM Cell Trace Violet (Invitrogen Molecular Probes). Cells were cultured for 48 h in DMEM complete media (DMEM with 10% FBS (Denville Scientific), 1% l-glutamine (Gibco), 1% penicillin–streptomycin (Gibco), 25 mM HEPES buffer, and 55 µM 2-β-mercaptoethanol (Sigma-Aldrich)) with 20 ng/ml rmIL-2, 20 ng/ml rmIL-7, 50 ng/ml rmIL-33 (all cytokines from R&D
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Systems) with DMSO or 500 µM nor-NOHA (Cayman Chemical) at 37 °C. Dilution of Cell Trace Violet was measured by flow cytometry.
RNA isolation and real-time quantitative PCR. For analysis from lung tissue, lungs were perfused as described above, and two small representative pieces from the right lobe were placed in RNAlater (Qiagen). Tissue was later homog-enized in RLT buffer, and RNA was isolated using an RNeasy mini kit according to the manufacturer’s instructions (Qiagen). For analysis of purified immune cells, Lin−CD45+CD90+CD25+IL-33R+ ILC2s or CD45+CD11c+F4/80+ mac-rophages were sort-purified from wild-type mice treated with 30 µg of papain for 5 d. Cells were sorted directly into TRIzol, and RNA was isolated via chlo-roform extraction. For both methods, cDNA was generated via Superscript reverse transcription (Invitrogen). Real-time quantitative PCR (qRT-PCR) was carried out on cDNA with SYBR Green master mix (Applied Biosystems) and commercially available primer sets (Qiagen). Reactions were run on a real-time PCR system (ABI7500, Applied Biosystems). Samples were normalized to β-actin, and results are shown as a fold induction relative to expression levels in PBS-treated or naive tissue as indicated.
Tissue histological sections. Left lobes of lungs were inflated, fixed with 4% paraformaldehyde (bioWORLD) and embedded in paraffin, and 5-µm sections were used for staining with hematoxylin and eosin or periodic acid–Schiff stain. Image acquisition was done with a Nikon Eclipse Ti microscope, a Nikon Digital Sight DS-Fi2 camera, and NIS-Elements AT version 4.2 acquisition soft-ware (Nikon). Adobe Photoshop (CS5, Adobe Systems Inc.) was used to adjust brightness and contrast only (changes were applied to the whole image).
Liquid chromatography–mass spectrometry. Metabolite pool analysis. C57BL/6J mice were treated with 100 ng of rmIL-33 (R&D Systems) i.n. every 3 d for 2–3 weeks. Lin−CD45+CD90+CD25+IL-33R+ ILC2s were sort- purified from the lung. 2 × 105 cells per well were cultured in a 96-well plate with DMEM complete media with 20 ng/ml rmIL-2, 20 ng/ml rmIL-7, 50 ng/ml rmIL-33 and DMSO or 500 µM nor-NOHA at 37 °C for 24 h. Samples were harvested into Eppendorf tubes, centrifuged at 5,000 r.p.m. for 5 min, and washed once with 1 ml of ice-cold PBS. Cell pellets were resuspended in 1 mL of 80% aqueous MeOH and stored at −80 °C to promote protein precipitation. Extracts were clarified by centrifugation at 21,000g for 20 min at 4 °C, dried using a Genevac benchtop evaporator (SP Industries) and reconstituted in 100 µL of 60:40 acetonitrile:water + 5 mM ammonium formate. Liquid chro-matography was performed using a 1290 HPLC system (Agilent Technologies) and an Xbridge BEH amide column (Waters Corporation; 150 mm × 2.1 mm, 3.5 µm). Samples were maintained at 4 °C with a 10-µL injection volume and 200 µL/min flow rate. The aqueous mobile phase (A) was 0.1% formic acid in water, and the organic mobile phase (B) was 0.1% formic acid in acetonitrile. Initial conditions were 95% B, and the following gradient program was applied: 1.0 min, 95% B; 9.0 min, 50% B; 12.0 min, 25% B; 13.0 min, 25% B; and 5-min re-equilibration time. Metabolites were detected with an Agilent 6550 Q-TOF with a JetStream source operating in positive mode with a gas temperature of 240 °C, a gas flow of 18 l/min, a nebulizer pressure of 25 psig, sheath gas conditions of 400 °C and 11 l/min, a capillary voltage of 4,000 V, fragmentor at
300 V and continuous reference mass correction at m/z 121.05087 and 922.00980. Metabolites were identified by the combination of accurate mass and retention time relative to authentic standards, and peak areas were extracted and integrated using Agilent MassHunter software.
Stable isotope tracing. C57BL/6J mice were treated with 100 ng of rmIL-33 i.n. every 3 d for 2–3 weeks. Lin−CD45+CD90+CD25+IL-33R+ ILC2s were sort-purified from the lung. 5 × 105 cells/ml were cultured in a 48-well plate with DMEM complete media with 20 ng/ml rmIL-2, 20 ng/ml rmIL-7, 50 ng/ml rmIL-33 and DMSO or 500 µM nor-NOHA at 37 °C for 36 h. Samples were centrifuged at 1,600 r.p.m. for 5 min and washed once with ice-cold PBS. Cells were then cultured at a concentration of 3.3 × 105 cells/ml in arginine-free, lysine-free, high-glucose DMEM media (Gibco, formulation A14431) supplemented with 10% dialyzed FBS (Gibco), 1% l-glutamine, 1% peni-cillin–streptomycin, 25 mM HEPES buffer, 55 µM 2-β-mercaptoethanol, 0.80 mM lysine and 0.4 mM U-13C-l-arginine (Cambridge Isotopes). Cells were collected by centrifugation at 5,000 r.p.m. for 5 min and prepared for LC-MS analysis as described above. Extracted ion chromatograms for all mass isotopomers were generated with Agilent MassHunter software and corrected for natural abundance with IsoCor software43.
Seahorse cellular metabolic profiling. Foxp3eGFP reporter mice were treated with 100 ng of rmIL-33 (R&D Systems) i.n. every 3 d for 2–3 weeks. Lin−CD45+CD90+CD25+IL-33R+ ILC2s and/or CD45+CD3+CD5+CD4+Foxp3−IL-33R+ TH2 cells were sort-purified from the lung and rested for 18 h in DMEM complete media (DMEM with 10% FBS (Denville Scientific), 1% l-glutamine (Gibco), 1% penicillin–streptomycin (Gibco), 25 mM HEPES buffer, and 55 µM 2-β-mercaptoethanol (Sigma-Aldrich)) on ice. For Arg1-inhibitor studies only, cells were then cultured for an additional 24 h in DMEM complete media with 20 ng/ml rIL-2, 20 ng/ml rIL-7 and 50 ng/ml rIL-33 (all cytokines from R&D Systems) at 37 °C. Cells were plated at 200,000 cells per well, and OCR and ECAR were measured in XF media (nonbuffered RPMI 1640 containing 25 mM glucose, 2 mM l-glutamine and 1 mM sodium pyruvate) under basal conditions, in response to 1 µM oligomycin, 1.5 µM fluoro-carbonyl cyanicide phenylhydrazone (FCCP) and 100 nM rotenone + 1 µM antimycin A (Sigma-Aldrich) and as indicated after injection with DMSO or 500 µM nor-NOHA using a 96-well extracellular flux analyzer (Seahorse Bioscience).
Statistical analysis. Results represent the mean ± s.e.m. unless otherwise indi-cated. Statistical significance was determined as indicated in figure legends. Statistical analyses were done with Prism GraphPad software v5.0. Researchers were not blinded to experimental groups. Exclusion criteria were not applied, as no experimental samples were excluded from analysis.
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49. Brestoff, J.R. et al. Group 2 innate lymphoid cells promote beiging of white adipose tissue and limit obesity. Nature 519, 242–246 (2015).
50. Osborne, L.C. et al. Virus-helminth coinfection reveals a microbiota-independent mechanism of immunomodulation. Science 345, 578–582 (2014).
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